PROJECT SUMMARY Macrophage fusion resulting in the formation of multinucleated giant cells (MGCs) accompanies a variety of maladies associated with chronic inflammation, including the foreign body response (FBR) elicited by implanted biomaterials. Despite the long history of research on FBR, the molecular and cellular mechanisms of macrophage fusion, an event central to the long-term failure of implanted prosthetic vascular grafts and other medical devices, remain poorly understood. In our preliminary studies using in vivo implantation model, we found that the formation of MGCs and granulation tissue, which develops around the implant and is a precursor of the undesirable fibrotic cap, was almost completely abolished in fibrinogen-deficient mice. Surprisingly, the number of MGCs formed on biomaterials implanted into Mac-1-deficient mice was greater than in wild-type mice and the thickness of granulation tissue was larger. We hypothesize that macrophage fusion on biomaterials critically depends on the deposited fibrin(ogen) matrix and the absence of Mac-1, through the alteration of adhesive properties of macrophages, exacerbates the FBR. Specific Aim 1 is to test this hypothesis. Using a mouse model of biomaterial implantation and gene-targeted mice, we will perform systematic analyses of the early and late stages of FBR and determine the M1/M2 phenotype of MGCs derived from wild-type and Mac-1-deficient macrophages. Using nanotechnology approaches we will characterize the adhesive and mechanical properties of fibrin(ogen) matrices deposited on biomaterials in wild-type and Mac-1-deficient mice. Specific Aim 2 will characterize previously unrecognized actin- based zipper-like structures (ZLS) that form between MGCs on implanted biomaterials. We developed an in vitro model that reproduces the formation of ZLS and demonstrated that the intercellular space within ZLS is filled with junctional proteins E-cadherin and nectin-2. We hypothesize that MGCs form epithelial-like junctions that aid the MGC survival. Taking advantage of technological innovations including a microfluidic chamber that allows the precise dissection of ZLS followed by proteomics analyses, high-resolution microscopy, live cell imaging and mice with myeloid cell-specific KO of E- cadherin and other components of junctions, we will determine the composition of ZLS and their role in the FBR. Specific Aim 3 is to determine the role of authentic fusogenic proteins syncytins in macrophage fusion. Based on our finding that macrophage fusion is initiated by an actin-based protrusion, we will use knockdown experiments, EM and video microscopy to test the hypothesis that a fusion-competent protrusion at the leading edge of a donor macrophage contains syncytins. Overalls, these studies will define the novel biology of macrophage fusion and characterize new mechanisms that have the potential to modulate the FBR.